Hypoxia is a common feature of solid tumors and predicts poor clinical outcome. Hypoxic tumor cells are resistant to radiation therapy and exhibit more aggressive phenotype. It has been suggested that hypoxic tumor cells possess undifferentiated characteristics and highly aggresive properties. Other evidence suggests that undifferentiated cells may potentially be more resistant to radiation than are differentiated cells, and recently, the hypoxia-inducible factor-1 (HIF-1) has been implicated in the regulation of radiation sensitivity of solid tumors. However, it remains unclear how hypoxia regulates tumor cell differentiation. We have begun to investigate the role of hypoxia in regulating cell differentiation using several model systems. Our data have shown that hypoxia inhibits cell differentiation and can arrest precursor/stem cells in their undifferentiated state. In our preliminary studies, we have aslo found that hypoxia inhibits differentiation of cancer cells and protects their clonogenic potential. These lines of evidence prompt us to hypothesize that hypoxia promotes tumor malignancy and resistance to radiation therapy, in part, by maintaining tumorigenic cells in their undifferentiated state. In this proposal, we will use neuroblastoma as a model to test this hypothesis by focusing on the following four working hypotheses. 1. Hypoxia inhibits differentiation of neuroblastoma cells in vitro. 2. Hypoxia maintains the poorly differentiated neuroblastoma cell population in hypoxic tumors. 3. The hypoxia inducible factor (HIF) regulates undifferentiated phenotype of neuroblastoma cells. 4. Hypoxia induces dedifferentiation of neuroblastoma. This proposal is innovative as it addresses the regulation of stem/precursor phenotype by tumor microenvironment, an important mechanism for tumor progression that has not yet been well understood. Our proposal will potentially provide new insight into how hypoxia increases tumor resistance to therapy and promotes malignant progression. Such insight could lead to new strategies to target the regulation of tumor cell differentiation as a form of cancer therapy.